Multisulfonated naphthalene ureas useful as complement inhibitors

ABSTRACT

Multisulfonated naphthalene ureas, useful as complement inhibitors.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of our co-pendingapplication, Ser. No. 413,938, filed Sept. 1, 1982, now abandoned, whichis a continuation-in-part of Ser. No. 334,941, filed Dec. 28, 1981,abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel multisulfonated naphthalene ureasand their use as inhibitors of the complement system of warm-bloodedanimals.

2. Description of the Prior Art

The term "complement" refers to a complex group of proteins in bodyfluids that, working together with antibodies or other factors, play animportant role as mediators of immune, allergic, immunochemical and/orimmunopathological reactions. The reactions in which complementparticipates take place in blood serum or in other body fluids, andhence are considered to be humoral reactions.

With regard to human blood, there are at present more than 20 proteinsin the complement system consisting of the so-called classical andalternative pathways. These complement proteins are generally designatedby the letter C and by number: C1, C2, C3 and so on up to C9. Thecomplement protein C1 is actually an assembly of subunits designatedC1q, C1r and C1s. The numbers assigned to the complement proteinsreflect the sequence in which they become active, with the exception ofcomplement protein C4, which reacts after C1 and before C2. Thenumerical assignments for the proteins in the complement system weremade before the reaction sequence was fully understood. A more detaileddiscussion of the complement system and its biochemical, biological andpathological role in the body processes can be found in, for example,Bull. W.H.O. 39:935 (1968); Annu. Rev. Med. 19:1 (1968); Johns HopkinsMed. J. 128:57 (1971); Harvey Lect. 66:75 (1972); N. Engl. J. Med.287:452, 489, 454, 592, 642 (1972); Sci. Am. 229 (5):54 (1973); Fed.Proc. 32:134 (1973):Med. World, Oct. 11, 1974, p. 53; J. Allergy Clin.Immunol. 53:298 (1974); Cold Spring Harbor Conf. Cell Proliferation2/Proteases Biol. Control:229 (1975); Annu. Rev. Biochem. 44: 697(1975); Complement in Clinical Medicine, Dis. Mon. (1975); Complement,Scope, December 1975; Ann. Intern. Med. 84:580 (1976); TransplantRev.:32 (1976); "Complement: Mechhanisms and Functions," Prentice-Hall,Englewood Cliffs, N.J. (1976); Essays Med. Biochem. 2:1 (1976); Hosp.Pract. 12:33 (1977); Perturbation of Complement in Disease, Chap. 15 inBiol. Amplification Systems in Immunol. (Ed. Day and Good), Plenum, N.Y.and London (1977); Am. J. Clin. Pathol. 68:647 (1977); Biochem. Soc.Trans. 5:1659 (1977); Harvey Lect. 72:139 (1976-1977); J. Periodontol.48:505 (1977); Biochem. Soc. Trans. 6:798 (1978); Clin. and Exp.Dermatol. 4:271 (1979); Infect. Dis. Rev. 1:483 (1979).

The complement system (e.g., classical pathway) can be considered toconsist of three subsystems: (1) a recognition unit (C1q) which enablesit to combine with antibody molecules that have detected a foreigninvader; (2) an activation unit (C1r, C1s, C2, C4, C3) which prepares asite on the neighboring membrane; and (3) an attack unit (C5, C6, C7, C8and C9) which creates a "hole" in the membrane. The membrane attack unitis nonspecific; it destroys invaders only because it is generated intheir neighborhood. In order to minimize damage to the host's own cells,its activity must be limited in time. This limitation is accomplishedpartly by the spontaneous decay of activated complement and partly byinterference by inhibitors and destructive enzymes. The control ofcomplement, however, is not perfect, and there are times when damage isdone to host's cells. Immunity is, therefore, a double-edged sword.

Activation of the complement system also accelerates blood clotting.This action comes about by way of the complement-mediated release of aclotting factor from platelets. The biologically active complementfragments and complexes become involved in reactions that damage thehost's cells. These pathogenic reactions can result in the developmentof immune-complex diseases. For example, in some forms of nephritis,complement damages the basal membrane of the kidney, resulting in theescape of protein from the blood into the urine. The diseasedisseminated lupus erythematosus belongs in this category; its symptomsinclude nephritis, visceral lesions and skin eruptions. The treatment ofdiphtheria or tetanus with the injection of large amounts of antitoxinsometimes results in serum sickness, an immune-complex disease.Rheumatoid arthritis also involves immune complexes. Like disseminatedlupus erythematosus, it is an autoimmune disease in which the diseasesymptoms are caused by pathological effects of the immune system in thehost's tissues. In summary, the complement system has been shown to beinvolved with inflammation, coagulation, fibrinolysis, antibody-antigenreactions and other metabolic processes.

In the presence of antibody-antigen complexes the complement proteinsare involved in a series of reactions which may lead to irreversiblemembrane damage if they occur in the vicinity of biological membranes.Thus, while complement constitutes a part of the body's defensemechanism against infection it also results in inflammation and tissuedamage in the immunopathological process. The nature of certaincomplement proteins, suggestion regarding the mode of complement bindingto bioiogical membranes and the manner in which complement effectsmembrane damage are discussed in Annu. Rev. Biochem. 38:389 (1969); J.Exp. Med. 141:724 (1975); J. Immunol. 116:1431 (1976); 119:1, 1195,1358, 1482 (1977); 120:1841 (1978); Immunochemistry 15:813 (1978); J.Biol. Chem. 254:9908 (1979).

A variety of substances have been disclosed as inhibiting the complementsystem, i.e., as complement inhibitors. For example, the compounds3,3'-ureylenebis[6-(2-amino-8-hydroxy-6-sulfo-1-naphthylazo)benzenesulfonicacid], tetrasodium salt (chlorazol fast pink), heparin and a sulphateddextran have been reported to have an anti-complementary effect, Br. J.Exp. Pathol. 33:327 (1952). German Pat. No. 2,254,893 or South AfricanPat. No. 727,923 discloses certain1-(diphenylmethyl)-4-(3-phenylally)piperazines useful as complementinhibitors. Other chemical compounds having complement inhibitingactivity are disclosed in, for example, J. Med. Chem. 12:415, 902, 1049,1053 (1969); Can. J. Biochem. 47:547 (1969); J. Immunol. 104:279 (1970);J. Immunol. 106:241 (1971); J. Immunol. 111:1061 (1973); Biochim.Biophys. Acta 317:539 (1973); Life Sci. 13:351 (1973); J. Immunol.113:584 (1974); Immunology 26:819 (1974); J. Med. Chem. 17:1160 (1974);Biochim. Biophys. Res. Comm. 67: 225 (1975); Ann. N.Y. Acad. Sci.256:441 (1975); J. Med. Chem. 19:634, 1079 (1976); J. Immunol. 118:466(1977); Arch. Int. Pharmacodyn. 226:281 (1977); Biochem. Pharmacol.26:325 (1977); J. Pharm. Sci. 66:1367 (1977); Chem. Pharm. Bull. 25:1202(1977); Biochim. Biophys. Acta 484:417 (1977); J. Clin. Microbiol. 5:278(1977); Immunochemistry 15:231 (1978); Immunology 34:509 (1978); J. Exp.Med. 147:409 (1978); Thromb. Res. 14:179 (1979); J. Immunol. 122:2418(1979); J. Chem. Soc. Chem. Comm. 726 (1979); Immunology 36:131 (1979);Biochim. Biophys. Acta 611:196 (1980); and J. Med. Chem. 23:240 (1980).

It has been reported that the known complement inhibitors,epsilon-aminocaproic acid and tranexamic acid, have been used withsuccess in the treatment of hereditary angioneurotic edema, a diseasestate resulting from an inherited deficiency or lack of function of theserum inhibitor of the activated first component of complement (C1inhibitor), N. Engl. J. Med. 286:808 (1972); 287:452 (1972); Ann.Intern. Med. 84:580 (1976); J. Allergy Clin. Immunol. 60:38 (1977). Alsoandrogenic steroids have been used successfully in the treatment of thisphysiological disorder; see Medicine 58: 321 1979); Arthritis Rheum.22:1295 (1979); Am. J. Med. 66:681 (1979); and J. Allergy Clin. Immunol.65:75 (1980).

It has also been reported that the drug pentosanpolysulfoester has ananticomplementary activity on human serum, both in vitro and in vivo, asjudged by the reduction in total hemolytic complement activity, Pathol.Biol. 25:33; 25 (2):105; 25 (3):179 (1977).

SUMMARY OF THE INVENTION

It has now been discovered that multisulfonated naphthalene ureasinteract with the complement reaction sequence, thereby inhibitingcomplement activity in body fluids.

This invention also concerns a method of inhibiting the complementsystem in a body fluid which comprises subjecting body fluid complementto the action of an effective complement inhibiting amount of theabove-identified compounds. This invention further deals with a methodof inhibiting the complement system in a warm-blooded animal whichcomprises administering to said animal an effective complementinhibiting amount of the above described compounds.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, there are provided novelcompounds represented by the following generic Formula I: ##STR1##wherein R₁ is hydrogen or --SO₃ A; R₂ is hydrogen, hydroxy, --SO₃ A orphenylsulfonyloxy; R₃ is hydrogen or --SO₃ A; R₄ is hydrogen, nitro,amino or --SO₃ A; R₅ is hydrogen or --SO₃ A; R₆ is hydrogen or --SO₃ A;R₇ is hydrogen or --SO₃ A; X is oxygen or sulfur; and A is a nontoxicpharmaceutically acceptable salt; provided that the naphthyl ring mustcontain at least one sulfonic acid group or no more than three sulfonicacid groups at the same time; and further provided that if R₂ is hydroxyand R₄ is --SO₃ A when X is oxygen, then at least one member selectedfrom R₁, R₃, R₅, R₆ or R₇ must be --SO₃ A.

Particularly preferred compounds of Formula I which are of maJorinterest as complement inhibitors include the following:

3,3'-Ureylenedi-2,7-naphthalenedisulfonic acid, tetrasodium salt.

3,3'-Ureylenebis(7-nitro-1,5-naphthalenedisulfonic acid), tetrasodiumsalt.

7,7'-Ureylenedi-1,3,5-naphthalenetrisulfonic acid, hexasodium salt.

3,3'-Ureylenebis(5-hydroxy-2,7-naphthalenedisulfonic acid), tetrasodiumsalt.

The instant invention also includes novel compounds represented by thefollowing generic Formula II: ##STR2## wherein R₁ is hydrogen or --SO₃A; R₂ is hydrogen, hydroxy, --SO₃ A or phenylsulfonyloxy; R₃ is hydrogenor --SO₃ A; R₄ is hydrogen, nitro, amino or --SO₃ A; R₅ is hydrogen or--SO₃ A; R₆ is hydrogen or --SO₃ A; R₇ is hydrogen or --SO₃ A; X isoxygen or sulfur; and A is a nontoxic pharmaceutically acceptable salt;provided that the naphthyl ring must contain at least one sulfonic acidgroup or no more than three sulfonic acid groups at the same time; andfurther provided that if R₂ is hydroxy and R₄ is --SO₃ A when X isoxygen, then at least one member selected from R₁, R₃, R₅, R₆ or R₇ mustbe --SO₃ A.

Particularly preferred compounds of Formula II which are of majorinterest as complement inhibitors include the following:

8,8'-Ureylenedi-1,3,6-naphthalenetrisulfonic acid, hexasodium salt

8,8'-(2-Thioureylene)di-1,3,6-naphthalenetrisulfonic acid, hexasodiumsalt

8,8'-Ureylenedi-1,3,5-naphthalenetrisulfonic acid, hexasodium salt

4,4'-Ureylenedi-1-naphthalenesulfonic acid, disodium salt

4,4'-Ureylenedi-2,7-naphthalenedisulfonic acid, tetrasodium salt

4,4'-Ureylenedi-2,6-naphthalenedisulfonic acid, tetrasodium salt

4,4'-Ureylenedi-1,6-naphthalenedisulfonic acid, tetrasodium salt

4,4'-Ureylenebis(5-hydroxy-2,7-naphthalenedisulfonic acid), tetrasodiumsalt dibenzenesulfonate

8,8'-Ureylenedi-1,6-naphthalenedisulfonic acid, tetrasodium salt

The present invention further concerns novel compounds represented bythe following generic Formula III: ##STR3## wherein R₁ is hydrogen or--SO₃ A; R₂ is hydrogen, --SO₃ A or phenylsulfonyloxy; R₃ is hydrogen or--SO₃ A; R₄ is hydrogen, nitro, amino or --SO₃ A; R₅ is hydrogen or--SO₃ A; R₆ is hydrogen or --SO₃ A; R₇ is hydrogen or --SO₃ A; X isoxygen or sulfur; and A is a nontoxic pharmaceutically acceptable salt;provided that the naphthyl ring must contain at least one sulfonic acidgroup or no more than three sulfonic acid groups at the same time.

A particularly preferred compound of Formula III which is of majorinterest as a complement inhibitor includes the following:

6,6'-Ureylenedi-1,3-naphthalenedisulfonic acid, tetrasodium salt

This invention is also concerned with known compounds prepared by Brassand co-workers as recited in Elsevier's Encyclopedia of Org. Chem., vol.12B at p. 5375 & p. 5386 (1955) and illustrated as Formulas IV and V:##STR4##

Specific known compounds of Formulas IV and V, respectively, which areof particular interest showing novel activity as complement inhibitorsare 7,7'-ureylenebis(1-hydroxy-3-naphthalenesulfonic acid),6,6'-ureylenebis(1-hydroxy-3-naphthalenesulfonic acid) and the disodiumsalts thereof.

This invention further deals with a method of inhibiting the complementsystem in a body fluid, such as blood serum, which comprises subjectingbody fluid complement to the action of an effective complementinhibiting amount of a compound of the above formulas. Body fluids caninclude blood, plasma, serum, synovial fluid, cerebrospinal fluid, orpathological accumulations of fluid such as pleural effusion, etc. Thisinvention also concerns a method of inhibiting the complement system ina warm-blooded animal which comprises administering to said warm-bloodedanimal an effective complement inhibiting amount of a compound of theabove formulas.

The above compounds of the present invention find utility as complementinhibitors in body fluids and as such may be used to ameliorate orprevent those pathological reactions tequiring the function ofcomplement and in the therapeutic treatment of warm-blooded animalshaving immunologic diseases such as rheumatoid arthritis, systemic lupuserythematosus, certain kinds of glomerulonephritis, certain kinds ofautoallergic hemolytic anemia, certain kinds of platelet disorders andcertain kinds of vasculitis. These compounds may also be used in thetherapeutic treatment of warm-blooded animals having non-immunologicdiseases such as paroxysmal nocturnal hemoglobinurea, hereditaryangioneurotic edema (such as Suramin Sodium, etc.) and inflammatorystates induced by the action of bacterial or lysosomal enzymes on theappropriate complement components as, for example, inflammationfollowing coronary occlusion. They may also be useful in the treatmentof transplant rejection and ulcers and as blood culture and transportmediums.

The compounds of the present invention may be prepared according to thefollowing flowcharts. ##STR5## wherein R₁, R₂, R₃, R₄, R₅, R₆ and R₇ areas above defined.

General Procedure A

Phosgene is bubbled into a vigorously stirred and cooled aqueoussolution of the amino acid (1) and sodium carbonate (at a ratio of 2-5moles of sodium carbonate per mole of amine) until the solution isacidic. The reaction is monitored by thin-layer electrophoresis orthin-layer chromatography. If required, more sodium carbonate is addedand phosgenation is repeated until the mixture is acidic. The mixture ismade weakly basic with sodium hydroxide or sodium carbonate, and ethanolis added to precipitate the product (2). The solid is collected byfiltration, washed successively with aqueous ethanol, ethanol and etherand dried by conventional procedures.

General Procedure B

An aqueous solution of the amino acid (1) and pyridine (at a ratio of5-9 moles of pyridine per mole of amine) is phosgenated as described inGeneral Procedure A and then made strongly basic with sodium hydroxide.The product is recovered as described in General Procedure A.

It is generally preferred that the respective product of each processstep, described hereinabove, is separated and/or isolated prior to itsuse as starting material for subsequent steps. Separation and isolationcan be effected by any suitable purification procedure such as, forexample, evaporation, crystallization, column chromatography, thin-layerchromatography, distillation, etc. Also, it should be appreciated thatwhen typical reaction conditions (e.g., temperatures, mole ratios,reaction times) have been given, the conditions which are both above andbelow these specified ranges can also be used, though generally lessconveniently.

The term "pharmaceutically acceptable salts" refers to those salts ofthe parent compound which do ot significantly or adversely affect thepharmaceutical properties (e.g. toxicity, effectiveness, etc.) of theparent compound. The salt forming moieties of the present inventionwhich are pharmaceutically acceptable include the alkali metals (e.g.,sodium, potassium, etc.) alkaline earth metals (e.g., calcium, etc.);ammonia; and substituted ammonia selected from the group consisting oftrialkylamine (C₁ -C₆), piperidine, pyrazine, alkanolamine (C₂ -C₆) andcycloalkylamine (C₃ -C₆).

The term "trialkylamine (C₁ -C₆)" defines those amines having threealiphatic fully saturated hydrocarbon substituents containing 1 to 6carbon atoms either linearly or branched. Typically, these amines aretrimethylamine, triethylamine, tripropylamine, dimethylamine,dimethyl-1-propylamine, etc. The term "alkanolamine (C₂ -C₆)" refers tothe above-defined trialkylamines additionally substituted with at leastone and not more than three hydroxy groups on at least two of the alkylhydrocarbon chains. Such amines are, for example, triethanolamine,tripropanolamine, etc. The term "cycloalkylamine (C₃ -C₆)" is defined asthe 3 to 6 fully saturated carbocyclic moieties such as cyclopropyl,methylcyclobutyl, cyclopentyl, cyclohexyl, etc.

As used hereinabove and below unless expressly stated to the contrary,all temperatures and temperature ranges refer to the centigrade systemand the terms "ambient" or "room temperature" refer to about 25° C. Theterm "percent" or "(%)" refers to weight percent and the terms "mole"and "moles" refer to gram moles. The term "equivalent" refers to aquantity of reagent equal in moles to the moles of the preceding orsucceeding reactant recited in the Preparation or Example in the term ofmoles of finite weight or volume.

Whereas the exact scope of the instant invention is set forth in theappended claims, the following specific examples illustrate certainaspects of the present invention. However, the examples are set forthfor illustration only and are not to be construed as limitations on thepresent invention except as set forth in the appended claims.

A further understanding of the invention can be obtained from thefollowing non-limiting Preparations and Examples.

EXAMPLE 1 2,2'-Ureylenedi-1,5-naphthalenedisulfonic acid, tetrasodiumsalt

A solution of 12.12 g of 2-naphthylamine-1,5-disulfonic acid in 75 ml ofwater and 20 ml of pyridine was treated with gaseous phosgene untilacidic. The solution was neutralized with pyridine, diluted with 500 mlof ethanol and made basic with 12.8 g of sodium hydroxide. The mixturewas stirred, then the precipitate was collected, dissolved in 100 ml of1N sodium hydroxide, evaporated in vacuo and cooled. The solid wascollected, washed with water and dried at 110° C., giving 8.02 g of thedesired product.

EXAMPLE 2 1,1'-Ureylenedi-2-naphthalenesulfonic acid

A solution of 8.93 g of 1-amino-2-naphthalenesulfonic acid in 100 ml ofwater and 16 ml of pyridine was phosgenated as described in Example 1,giving 2.5 g of the desired product.

EXAMPLE 3 4,4'-Ureylenebis(5-hydroxy-2,7-naphthalenedisulfonic acid),tetrasodium salt dibenzenesulfonate

A solution of 4.95 g of4-amino-5-hydroxybenzenesulfonate-2,7-naphthalenedisulfonic acid in 40ml of water and 7.5 ml of pyridine was phosgenated as described inExample 1, giving 200 mg of the desired product.

EXAMPLE 4 8,8'-Ureylenedi-1,3,6-naphthalenetrisulfonic acid, hexasodiumsalt

A solution of 51.2 g of 8-amino-1,3,6-naphthalenetrisulfonic aciddisodium salt in 120 ml of water containing 21.0 ml of 5N sodiumhydroxide was warmed and filtered. The filtrate was slowly diluted with400 ml of ethanol, stirred and allowed to cool to room temperature. Thesolid was collected, washed with ethanol, then ether and dried, giving46.0 g of 8-amino-1,3,6-naphthalenetrisulfonic acid, trisodium salt.

A solution of 2.0 g of 8-amino-1,3,6-naphthalenetrisulfonic acid,trisodium salt and 0.93 g of sodium carbonate in 15 ml of water wasphosgenated until acidic, neutralized with 150 mg of sodium carbonate,heated on a steam bath and diluted with 30 ml of ethanol. The solid wascollected, washed with aqueous ethanol (1:2), ethanol, then ether anddried, giving 1.5 g of the desired product.

EXAMPLE 5 3,3'-Ureylenedi-2,7-naphthalenedisulfonic acid, tetrasodiumsalt

A solution of 13.01 g of 3-amino-2,7-naphthalenedisulfonic acid,monosodium salt and 21 g of sodium carbonate in 75 ml of water wasphosgenated as described in Example 4, giving 3.61 g of the desiredproduct.

EXAMPLE 6

5,5'-Ureylenedi-1-naphthalenesulfonic acid, disodium salt

A solution of 8.93 g of 5-amino-1-naphthalenesulfonic acid and 21 g ofsodium carbonate in 100 ml of water was phosgenated as described inExample 4, giving 1.83 g of the desired product.

EXAMPLE 7

8,8'-(2-Thioureylene)di-1,3,6-naphthalenetrisulfonic acid, hexasodiumsalt

To a solution of 32.0 g of 8-amino-1,3,6-naphthalenetrisulfonic acid,trisodium salt in 350 ml of water and 7.0 ml of concentratedhydrochloric acid was added 10.0 g of thiophosgene. The mixture wasstirred 21/2 hours, treated with charcoal and filtered throughdiatomaceous earth. The filtrate was neutralized with 46 ml of 5N sodiumhydroxide, chilled and the solid was collected and recrystallized from40 ml of water by heating and then chilling in an ice bath. This solidwas washed with ice water, acetone then ether and dried, giving 11.2 gof 8-isothiocyanato-1,3,6-naphthalenetrisulfonic acid, trisodium salt.

A solution of 5.0 g of 8-isothiocyanato-1,3,6-naphthalenetrisulfonicacid, trisodium salt in 20 ml of water was heated on a steam bath forone hour, then cooled and diluted with 40 ml of ethanol. The solid wascollected, washed with 66% ethanol, ethanol then ether and dried, giving4.15 g of the desired product.

EXAMPLE 8 4,4'-Ureylenedi-2,6-naphthalenedisulfonic acid, tetrasodiumsalt

A suspension of 15.15 g of 1-amino-3,7-naphthalenedisulfonic acid, 20 mlof 5N sodium hydroxide and 14.6 g of sodium carbonate in 150 ml of waterwas phosgenated as described in Example 4, giving 7.0 g of the desiredproduct.

EXAMPLE 9 4,4'-Ureylenedi-1,6-naphthalenedisulfonic acid, tetrasodiumsalt

A solution 15.15 g of 1-amino-4,7-naphthalenedisulfonic acid, 20.0 ml of5N sodium hydroxide and 10.6 g of sodium carbonate in 100 ml of waterwas phosgenated as described in Example 4, giving 6.8 g of the desiredproduct.

EXAMPLE 10 8,8'-Ureylenedi-1,6-naphthalenedisulfonic acid, tetrasodiumsalt

A solution of 15.15 g of 1-amino-3,8-naphthalenedisulfonic acid, 14.6 gof sodium carbonate, 20.0 ml of 5N sodium hydroxide and 50 ml of waterwas phosgenated as described in Example 4, giving 13.8 g of the desiredproduct.

EXAMPLE 11 3,3'-Ureylenebis(7-nitro-1,5-naphthalenedisulfonic acid),tetrasodium salt

A 100 g portion of 3-amino-7-nitro-1,5-naphthalenedisulfonic acid wasslurried in 800 ml of water, the pH was adjusted to 8-9, the mixture wasconcentrated until a solid began to precipitate and then allowed tostand overnight. The solid was collected, washed with cold water,ethanol then ether and dried, giving 34 g of3-amino-7-nitro-1,5-naphthalenedisulfonic acid, disodium salt.

A 7.84 g portion of the above product and 8.4 g of sodium carbonate inwater was phosgenated as described in Example 4, giving 5 g of thedesired product.

EXAMPLE 12 7,7'-Ureylenedi-1,3,5-naphthalenetrisulfonic acid, hexasodiumsalt

A 450 g portion of 2-amino-6-nitro-4,8-naphthalenedisulfonic acid wasdissolved in 2.3 liters of water and basified to pH 9 with 5N sodiumhydroxide. After standing 48 hours, the solid was collected, washed withethanol and dried, giving 193 g of2-amino-6-nitro-4,8-naphthalenedisulfonic acid, disodium salt.

To a heavy paste of 78.0 g of the above disodium salt in water was added15.0 g of sodium nitrite. This mixture was then slowly added to a cooledsolution of 80.0 ml of concentrated hydrochloric acid in 40 ml of water,maintaining the temperature at 0°-5° C. A solution of 4.6 g of cupricchloride in 4 ml of water was added to a solution of 200 ml of glacialacetic acid saturated with sulfur dioxide. This mixture was added to theabove diazonium salt mixture slowly, maintaining the temperature at5°-7° C. Additional water was introduced to aid stirring. The mixturewas stirred for 2 hours then stored in a chill room for 48 hours. Thesolvent was removed, the residue was dissolved in water, basified to pH9-10 with sodium carbonate and filtered. The filtrate was concentrated,then diluted with ethanol and the solid was collected, washed withethanol and ether and dried, giving 70.0 g of3-nitro-1,5,7-naphthalenetrisulfonic acid, trisodium salt. A 26 gportion of this trisodium salt was catalytically reduced giving 12 g of3-amino-1,5,7-naphthalenetrisulfonic acid, trisodium salt. A 5.0 gportion of this amino derivative and 2.5 g of sodium bicarbonate inwater was phosgenated as described in Example 4, giving 4.3 g of thedesired product.

EXAMPLE 13 6,6'-Ureylenedi-1,3-naphthalenedisulfonic acid, tetrasodiumsalt

A mixture of 6.06 g of 6-amino-1,3-naphthalenedisulfonic acid and 10.5 gof sodium carbonate in 50 ml of water was phosgenated as described inExample 4, giving 7.2 g of the desired product.

EXAMPLE 14 4,4'-Ureylenedi-1-naphthalenesulfonic acid, disodium salt

A mixture of 10.17 g of 4-amino-1-napthalenesulfonic acid and 8.40 g ofsodium carbonate in 100 ml of water was phosgenated as described inExample 4, giving 7.81 g of the desired product.

EXAMPLE 15 4,4'-Ureylenedi-2,7-naphthalenedisulfonic acid, tetrasodiumsalt

A mixture of 12.12 g of 4-amino-2,7-naphthalenedisulfonic acid and 21 gof sodium bicarbonate in 75 ml of water was phosgenated as described inExample 4, giving 14.41 g of the desired product.

EXAMPLE 16 8,8'-Ureylenedi-1,3,5-naphthalenetrisulfonic acid, hexasodiumsalt

To a warm solution of 23.8 g of 8-amino-1,3,5-naphthalenetrisulfonicacid in 25 ml of water and 25 ml of 5N sodium hydroxide was slowly addedwith vigorous stirring 125 ml of ethanol. The mixture was cooled to roomtemperature and the solid was collected, washed with 80% ethanol,ethanol then ether and dried, giving 21.0 g of8-amino--1,3,5-naphthalenetrisulfonic acid, trisodium salt.

A 4.35 g portion of the above trisodium salt and sodium carbonate in 15ml of water was phosgenated as described in Example 4, giving 1.85 g ofthe desired product.

EXAMPLE 17 3,3'-Ureylenebis(5-hydroxy-2,7-naphthalenedisulfonic acid),tetrasodium salt

A mixture of 7.25 g of 3-amino-5-hydroxy-2,7-naphthalenedisulfonic acid,disodium salt and 5.1 g of sodium bicarbonate in 75 ml of water wasphosgenated as described in Example 4, giving 1.6 g of the desiredproduct.

EXAMPLE 18 Preparation of Compressed Tablet

    ______________________________________                                        Ingredient          mg/Tablet                                                 ______________________________________                                        Active Compound      0.5-500                                                  Dibasic Calcium Phosphate NF                                                                      qs                                                        Starch USP          40                                                        Modified Starch     10                                                        Magnesium Stearate USP                                                                            1-5                                                       ______________________________________                                    

EXAMPLE 19 Preparation of Compressed Tablet - Sustained Action

    ______________________________________                                        Ingredient          mg/Tablet                                                 ______________________________________                                        Active Compound as Aluminum                                                                       0.5-500 (as acid                                          Lake*, Micronized   equivalent)                                               Dibasic Calcium Phosphate NF                                                                      qs                                                        Alginic Acid        20                                                        Starch USP          35                                                        Magnesium Stearate USP                                                                            1-10                                                      ______________________________________                                         *Complement inhibitor plus aluminum sulfate yields aluminum complement        inhibitor. Complement inhibitor content in aluminum lake ranges from          5-30%.                                                                   

EXAMPLE 20 Preparation of Hard Shell Capsule

    ______________________________________                                        Ingredient        mg/Capsule                                                  ______________________________________                                        Active Compound   0.5-500                                                     Lactose, Spray Dried                                                                            qs                                                          Magnesium Stearate                                                                               1-10                                                       ______________________________________                                    

EXAMPLE 21 Preparation of Oral Liquid (Syrup)

    ______________________________________                                        Ingredient        % W/V                                                       ______________________________________                                        Active Compound   0.05-5                                                      Liquid Sugar      75.0                                                        Methyl Paraben USP                                                                              0.18                                                        Propyl Paraben USP                                                                              0.02                                                        Flavoring Agent   qs                                                          Purified Water qs ad                                                                            100.0                                                       ______________________________________                                    

EXAMPLE 22 Preparation of Oral Liquid (Elixir)

    ______________________________________                                        Ingredient        % W/V                                                       ______________________________________                                        Active Compound   0.05-5                                                      Alcohol USP       12.5                                                        Glycerin USP      45.0                                                        Syrup USP         20.0                                                        Flavoring Agent   qs                                                          Purified Water qs ad                                                                            100.0                                                       ______________________________________                                    

EXAMPLE 23 Preparation of Oral Suspension (Syrup)

    ______________________________________                                        Ingredient         % W/V                                                      ______________________________________                                        Active Compound as Aluminum                                                                      0.05-5                                                     Lake, Micronized   (acid equivalent)                                          Polysorbate 80 USP 0.1                                                        Magnesium Aluminum Silicate,                                                                     0.3                                                        Colloidal                                                                     Flavoring Agent    qs                                                         Methyl Paraben USP 0.18                                                       Propyl Paraben USP 0.02                                                       Liquid Sugar       75.0                                                       Purified Water qs ad                                                                             100.0                                                      ______________________________________                                    

EXAMPLE 24 Preparation of Injectable Solution Ingredient

    ______________________________________                                        Ingredient       % W/V                                                        ______________________________________                                        Active Compound  0.05-5                                                       Benzyl Alcohol NF                                                                               0.9                                                         Water for Injection                                                                            100.0                                                        ______________________________________                                    

EXAMPLE 25 Preparation of Injectable Oil

    ______________________________________                                        Ingredient       % W/V                                                        ______________________________________                                        Active Compound  0.05-5                                                       Benzyl Alcohol    1.5                                                         Sesame Oil qs ad 100.0                                                        ______________________________________                                    

EXAMPLE 26 Preparation of Intra-Articular Product

    ______________________________________                                        Ingredient           Amount                                                   ______________________________________                                        Active Compound      2-20 mg                                                  NaCl (physiological saline)                                                                        0.9%                                                     Benzyl Alcohol       0.9%                                                     Sodium Carboxymethylcellulose                                                                      1.5%                                                     pH adjusted to 5.0-7.5                                                        Water for Injection qs ad                                                                          100%                                                     ______________________________________                                    

EXAMPLE 27 Preparation of Injectable Depo Suspension

    ______________________________________                                        Ingredient         % W/V                                                      ______________________________________                                        Active Compound    0.05-5                                                                        (acid equivalent)                                          Polysorbate 80 USP 0.2                                                        Polyethylene Glycol 4000 USP                                                                     3.0                                                        Sodium Chloride USP                                                                              0.8                                                        Benzyl Alcohol NF  0.9                                                        HCl to pH 6-8      qs                                                         Water for Injection qs ad                                                                        100.0                                                      ______________________________________                                    

EXAMPLE 28 Preparation of Dental Paste

    ______________________________________                                        Ingredient           % W/W                                                    ______________________________________                                        Active Compound      0.05-5                                                   Zinc Oxide           15                                                       Polyethylene Glycol 4000 USP                                                                       50                                                       Distilled Water qs   100                                                      ______________________________________                                    

EXAMPLE 29 Preparation of Dental Ointment

    ______________________________________                                        Ingredient         % W/W                                                      ______________________________________                                        Active Compound    0.05-5                                                     Petrolatum, White USP qs                                                                         100                                                        ______________________________________                                    

EXAMPLE 30 Preparation of Dental Cream

    ______________________________________                                        Ingredient          % W/W                                                     ______________________________________                                        Active Compound     0.05-5                                                    Mineral Oil         50                                                        Beeswax             15                                                        Sorbitan Monostearate                                                                              2                                                        Polyoxyethylene 20 Sorbitan                                                                        3                                                        Monostearate                                                                  Methyl Paraben USP    0.18                                                    Propyl Paraben USP    0.02                                                    Distilled Water qs  100                                                       ______________________________________                                    

EXAMPLE 31 Preparation of Topical Cream

    ______________________________________                                        Ingredient        % W/W                                                       ______________________________________                                        Active Compound   0.05-5                                                      Sodium Lauryl Sulfate                                                                            1                                                          Propylene Glycol  12                                                          Stearyl Alcohol   25                                                          Petrolatum, White USP                                                                           25                                                          Methyl Paraben USP                                                                                0.18                                                      Propyl Paraben USP                                                                                0.02                                                      Purified Water qs 100                                                         ______________________________________                                    

EXAMPLE 32 Preparation of Topical Ointment

    ______________________________________                                        Ingredient         % W/W                                                      ______________________________________                                        Active Compound    0.05-5                                                     Cholesterol        3                                                          Stearyl Alcohol    3                                                          White Wax          8                                                          Petrolatum, White USP qs                                                                         100                                                        ______________________________________                                    

EXAMPLE 33 Preparation of Spray Lotion (Non-aerosol)

    ______________________________________                                        Ingredient         % W/W                                                      ______________________________________                                        Active Compound    0.05-5                                                     Isopropyl Myristate                                                                               20                                                        Alcohol (Denatured) qs                                                                           100                                                        ______________________________________                                    

EXAMPLE 34 Preparation of Buccal Tablet

    ______________________________________                                        Ingredient          mg/Tablet                                                 ______________________________________                                        Active Ingredient   3.25                                                      6 × Sugar     290.60                                                    Acacia              14.53                                                     Soluble Starch      14.53                                                     F. D. & C. Yellow No. 6 Dye                                                                       0.49                                                      Magnesium Stearate  1.60                                                                          325.00                                                    ______________________________________                                    

The final tablet will weight about 325 mg and may be compressed intobuccal tablets in flat faced or any other tooling shape convenient forbuccal administration.

EXAMPLE 35 Preparation of Lozenge

    ______________________________________                                        Ingredient           g/Lozenge                                                ______________________________________                                        Active Ingredient    0.0140                                                   Kompact ® Sugar (Sucrest Co.)                                                                  0.7138                                                   6 × Sugar      0.4802                                                   Sorbitol (USP Crystalline)                                                                         0.1038                                                   Flavor               0.0840                                                   Magnesium Stearate   0.0021                                                   Dye                  qs                                                       Stearic Acid         0.0021                                                                        1.4000                                                   ______________________________________                                    

The ingredients are compressed into 5/8" flat based lozenge tooling.Other shapes may also be utilized.

The compounds of the present invention may be administered internally,e.g., orally, intra-articularly or parenterally, to a warm-bloodedanimal to ihhibit complement in the body fluid of the animal, suchinhibition being useful in the amelioration or prevention of thosereactions dependent upon the fuction of complement, such as inflammatoryprocess and cell membrane damage induced by antigen-antibody complexes.A range of doses may be employed depending on the mode ofadministration, the condition being treated and the particular compoundbeing used. For example, for intravenous or subcutaneous use from about5 to about 50 mg/kg/day, or every six hours for more rapidly excretedsalts, may be used. For intra-articular use for large joints such as theknee, from about 2 to about 20 mg/joint per week may be used, withproportionally smaller doses for smaller joints. The dosage range is tobe adjusted to provide optimum therapeutic response in the warm-bloodedanimal being treated. In general, the amount of compound administeredcan vary over a wide range to provide from about 5 mg/kg to about 100mg/kg of body weight of animal per day. The usual daily dosage for a 70kg subject may vary from about 350 mg to about 3.5 g. Unit doses of theacid or salt can contain from about 0.5 mg to about 500 mg.

The compounds of the present invention may also be administeredtopically in the form of ointments, creams, lotions and the like,suitable for the treatment of complement dependent dermatologicaldisorders.

Moreover, the compounds of the present invention may be administered inthe form of dental pastes, ointments, buccal tablets and othercompositions suitable for application periodontally for the treatment ofperiodontitis and related diseases of the oral cavity.

In therapeutic use, the compounds of this invention may be administeredin the form of conventional phatarmaceutial compositions. Suchcompositions may be formulated so as to be suitable for oral orparenteral administration. The active ingredient may be combined inadmixture with a pharmaceutically acceptable carrier, which carrier maytake a wide variety of forms depending on the form of preparationdesired for administration, i.e., oral or parenteral. The compounds canbe used in compositions such as tablets. Here the principal activeingredient is mixed with conventional tabletting ingredients such ascorn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesiumstearate, dicalcium phosphate, gums, or similar materials as nontoxicpharmaceutically acceptable diluents or carriers. The tablets or pillsof the novel compositions can be laminated or otherwise compounded toprovide a dosage form affording the advantage of prolonged or delayedaction or predetermined successive action of the enclosed medication.For example, the tablet or pill can comprise an inner dosage and anouter dosage component, the latter being in the form of an envelope overthe former. The two components can be separated by an enteric layerwhich serves to resist disintegration in the stomach and permits theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, Such matetials including a number of polymeric acids ormixtures of polymeric acids with such materials as shellac, shellac andcetyl alcohol, cellulose acetate and the like. A particularlyadvantageous enteric coating comprises a styrene maleic acid copolymertogether with known materials contributing to the enteric properties ofthe coating. The tablet or pill may be colored through the use of anappropriate nontoxic dye, so as to provide a pleasing appearance.

The liquid forms in which the novel compositions of the presentinvention may be incorporated for administration include suitableflavored emulsions with edible oils, such as, cottonseed oil, sesameoil, coconut oil, peanut oil, and the like, as well as elixirs andsimiiar pharmaceutical vehicles. Sterile suspensions or solutions can beprepared for parenteral use. Isotonic preparations containing suitablepreservatives are also desirable for injection use.

The term "dosage form," as described herein, refers to physicallydiscrete units suitable as unitary dosage for warm-blooded animalsubjects, each unit containing a predetermined quantity of activecomponent calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical diluent, carrier orvehicle. The specification for the novel dosage forms of this inventionis indicated by characteristics of the active component and theparticular therapeutic effect to be achieved or the limitations inherentin the art of compounding such an active component for therapeutic usein warm-blooded animals as disclosed in this specification. Examples ofsuitable oral dosage forms in accord with this invention are tablets,capsules, pills, powder packets, granules, wafers, cachets,teaspoonfuls, dropperfuls, ampules, vials, segregated multiples of anyof the foregoing and other forms as herein described.

The complement inhibiting activity of the compounds of this inventionhas been demonstrated by one or more of the following identified tests:(i) Test Code 026 (C1 inhibitor)--This test measures the ability ofactivated human C1 to destroy fluid phase human C2 in the presence of C4and appropriate dilutions of the test compound. An active inhibitorprotects C2 from C1 and C4; (ii) Test Code 036 (C-Shunt inhibitor)--Inthis test human erythrocytes rendered fragile are lysed in autologousserum via the shunt pathway activated by cobra venom factor in thepresence of appropriate dilutions of the test compound. Inhibition ofthe shunt pathway results in failure of lysis; (iii) Cap 50 Test--Here,appropriate amounts of the test compound are added to a pool of guineapig serum in vitro, after which the complement level is determined inundiluted serum by the serum capillary tube assay of U.S. Pat. No.3,876,376. The concentration of compound inhibiting 50% is reported; and(iv) Guinea Pig Intraperitoneal Test (GPIP)--Guinea pigs weighing about300 g are dosed intraperitoneally (i.p.) with 200 mg/kg of the testcompound dissolved in saline and adjusted to pH 7-8. Approximately 0.4ml blood samples, taken by orbital sinus puncture 30 minutes and onehour after injections, are collected directly into centrifuge tubes; 5ml blood samples, taken by decapitation 2 hours after inJection, arecollected directly into diSPo® beakers. The samples were allowed toclot, centrifuged, and the resultant sera were assayed for complementactivity using the capillary complement assay. Percent inhibition iscalculated by comparison with simultaneous controls. The results of theGPIP appear in Table I together with results of Test Code 026, 036 andCap 50. Table I shows that the principal compounds of the inventionpossess highly significant complement inhibiting activity inwarm-blooded animals.

                                      TABLE I                                     __________________________________________________________________________    Biological Activities                                                                                                  In vivo Activity                                                In vitro Activity                                                                           Guinea Pigs                                                          C-Shunt  Intraperitoneal                                                 Cl   Inhibition                                                                             % Lowering                                                      026* 036* Cap Time (minutes)                       Compound                   Wells                                                                              Wells                                                                              50  30 60                                                                              120                             __________________________________________________________________________    7,7'-Ureylenebis(1-hydroxy-3-naphthalenesulfonic acid)                                                    +2**                                              6,6'-Ureylenebis(1-hydroxy-3-naphthalenesulfonic acid)                                                   +1                                                 disodium salt                                                                 8,8'-Ureylenedi-1,3,6-naphthalenetrisulfonic acid, hexa-                                                 +5         180                                                                              25 61                                                                              65                              sodium salt                                                                   6,6'-Ureylenedi-1,3-naphthalenedisulfonic acid, tetra-                                                   +3                                                 sodium salt                                                                   4,4'-Ureylenedi-1-naphthalenesulfonic acid, disodium salt                                                +2                                                 3,3'-Ureylenedi-2,7-naphthalenedisulfonic acid, tetra-                                                   +3                                                 sodium salt                                                                   8,8'-(2-Thioureylene)di-1,3,6-naphthalenetrisulfonic acid,                                               +5         155                                                                              41 20                                                                              42                              hexasodium salt                                                               4,4'-Ureylenedi-2,7-naphthalenedisulfonic acid, tetra-                                                   +3        >500                                     sodium salt                                                                   8,8'-Ureylenedi-1,3,5-naphthalenetrisulfonic acid, hexa-                                                             12                                                                              92 92                                                                              94                              sodium salt                                                                   4,4'-Ureylenedi-1,6-naphthalenedisulfonic acid, tetra-                                                   +3        >500                                     sodium salt                                                                   4,4'-Ureylenedi-2,6-naphthalenedisulfonic acid, tetra-                                                   +2                                                 sodium salt                                                                   4,4'-Ureylenebis(5-hydroxy-2,7-naphthalenedisulfonic                                                     +3        >500                                     acid), tetrasodium salt dibenzenesulfonate                                    8,8'-Ureylenedi-1,6-naphthalenedisulfonic acid, tetra-                                                   +3        >500                                     sodium salt                                                                   3,3'-Ureylenebis(7-nitro-1,5-naphthalenedisulfonic acid),                                                +3        >500                                     tetrasodium salt                                                              7,7'-Ureylenedi-1,3,5-naphthalenetrisulfonic acid, hexa-                                                 +6   +1    280                                     sodium salt                                                                   3,3'-Ureylenebis(5-hydroxy-2,7-naphthalenedisulfonic                                                     +4          90                                                                               4  1                                                                              28                              acid), tetrasodium salt                                                       __________________________________________________________________________     *Tests identified by code herein.                                             **Activity in wells, a serial dilution assay; higher well number indicate     higher activity. The serial dilutions are twofold.                       

We claim:
 1. The compound 3,3'-ureylenedi-2,7-naphthalenedisulfonicacid, tetrasodium salt.
 2. The compound3,3'-ureylenebis(7-nitro-1,5-naphthalenedisulfonic acid), tetrasodiumsalt.
 3. The compound 7,7'-ureylenedi-1,3,5-naphthalenetrisulfonic acid,hexasodium salt.
 4. The compound,8,8'-ureylenedi-1,3,6-naphthalenetrisulfonic acid, hexasodium salt. 5.The compound, 8,8'-(2-thioureylene)di-1,3,6-naphthalenetrisulfonic acid,hexasodium salt.
 6. The compound,4,4'-ureylenedi-2,6-naphthalenedisulfonic acid, tetrasodium salt.
 7. Thecompound, 4,4'-ureylenedi-1,6-naphthalenedisulfonic acid, tetrasodiumsalt.
 8. The compound, 8,8'-ureylenedi-1,6-naphthalenedisulfonic acid,tetrasodium salt.
 9. The compound,8,8'-ureylenedi-1,3,5-naphthalenetrisulfonic acid, hexasodium salt. 10.The compound, 4,4'-ureylenedi-2,7-naphthalenedisulfonic acid,tetrasodium salt.
 11. The compound,6,6'-ureylenedi-1,3-naphthalenedisulfonic acid, tetrasodium salt.